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为了探讨小凹蛋白-1(caveolin-1,Cav-1)在人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)细胞外钙敏感受体(extracellular Ca2+-sensing receptor,CaR)介导Ca2+内流中的作用,本实验研究了细胞膜穴样凹陷(caveolae)结构破坏剂Filipin或Cav-1基因沉默后对CaR介导Ca2+内流的影响。Fura-2/AM负载检测细胞内Ca2+浓度(intracellular Ca2+ concentration,[Ca2+]i)。结果显示,HUVECs中CaR对不同浓度细胞外Ca2+刺激无反应。无论细胞外为零钙液或含钙液时,精胺(Spermine,2mmol/L)刺激CaR时均引起[Ca2+]i升高(P<0.05),其中细胞外液为含钙液时,[Ca2+]i升高较细胞外为零钙液时更明显(P<0.05),CaR的负性变构调节剂Calhex231(1μmol/L)均可完全阻断Spermine刺激引起的[Ca2+]i升高(P<0.05);相反,Spermine升高[Ca2+]i作用可被Filipin(1.5μg/mL)或Cav-1基因沉默进一步加强(均P<0.05)。免疫荧光技术检测显示HUVECs中有CaR和Cav-1表达,两者共定位于膜上。Western blot检测结果显示Cav-1干扰后,Cav-1蛋白表达降低,同时CaR的膜蛋白表达也降低(P<0.05),CaR的总蛋白表达无变化(P>0.05)。上述结果表明:Cav-1和CaR共定位于HUVECs膜,Cav-1对CaR介导的Ca2+内流有下调作用,其机制可能与Cav-1影响CaR膜定位及减弱其对激动剂反应性有关。
To investigate the effect of caveolin-1 (Cav-1) on Ca2 + mediated by extracellular Ca2 + -sensing receptor (CaR) in human umbilical vein endothelial cells (HUVECs) In this study, we investigated the effect of Filipin or Cav-1 gene silencing Ca2 + -mediated Ca2 + influx in caveolae structural disruptors. Fura-2 / AM load detection of intracellular Ca2 concentration (intracellular Ca2 concentration, [Ca2] i). The results showed that CaR in HUVECs did not respond to different concentrations of extracellular Ca2 + stimulation. Ca2 +] i increased (P <0.05) when Ca2 + was stimulated by Spermine (2mmol / L), and no extracellular Ca2 + or Ca2 + Ca2 +] i increased more significantly than that of extracellular calcium (P <0.05). Calhex231 (1μmol / L), a negative allosteric modulator of CaR, completely blocked the increase of [Ca2 +] i induced by Spermine stimulation (P <0.05). On the contrary, the increase of [Ca2 +] i by Spermine could be further enhanced by Filipin (1.5μg / mL) or Cav-1 gene silencing (all P <0.05). Immunofluorescence showed that CaR and Cav-1 were expressed in HUVECs, both of which were located on the membrane. The results of Western blot showed that the expression of Cav-1 protein was decreased and the expression of CaR protein was also decreased (P <0.05). The protein expression of CaR did not change after Cav-1 interference (P> 0.05). The results indicated that Cav-1 and CaR co-localized in HUVECs membrane, and Cav-1 had a down-regulation of CaR-mediated Ca2 + influx. The mechanism may be related to Cav-1 affecting CaR membrane localization and decreasing its responsiveness to agonists .